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Presented by : Priyanjali Ray

Regn. No. : 11408031

Semester 7

Department of Genetic Engineering.

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Introduction

What is Porphyria?

Also called Vampire Syndrome, is a group of rare disorders passeddown through families, in which an important part of Hemoglobin,called heme, is not made properly.

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What is Heme?

A prosthetic group that consists of an iron atom contained in the centerof a large heterocyclic organic ring called a porphyrin.

A part of Hemoglobin, which carries Oxygen to different parts of thebody.

Produced in the bone marrow and liver through a complex processcontrolled by eight different enzymes.

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Why does the disease occur?

I. During production of Heme, different intermediate compounds orprecursors are created and modified. If one of the eight essentialenzymes in Heme production is deficient due to mutation in thegene coding for it, certain precursors may accumulate in bonemarrow or liver, appear in excess in the blood, and get excreted in the

urine or stool. This may lead to various physical symptoms or evendamage of the nervous system.

II. Mutation causing the reduction in enzyme activity also limits theamount of heme being produced. This may result in degenerative

changes in the central nervous system. It also results in a decrease ofthe amount of Oxygen being sent to the heart for pumping andsubsequently leads to reduction in the amount of oxygen beingsupplied to various parts of the body.

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Heme Biosynthesis PathwayGlycine + Succinyl coA

ALA Synthase

Delta-Aminolaevulinic acid (ALA)

ALA Dehydratase

Porphobilinogen

Hydroxymethylbilane

Uroporphyrinogen III

Coproporphyrinogen III

Protoporphyrinogen IX

Protoporphyrin IX

Heme

Heme

PorphobilinogenDeaminase

UroporphyrinogenIIIsynthase

UroporphyrinogenDecarboxylase

CoproporphyrinogenOxidase

ProtoporphyrinogenOxidase

Ferrochelatase

ALAD

HMBS

Gene : ALAS2

UROS

UROD

CPOX

PPOX

FECH

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Where does Heme biosynthesis occur in the cell?

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Culture and History

Originally studied by Hippocrates but biochemically explained by Dr.Felix Hoppe-Seyler in 1874.

detected in all races, multiple ethnic groups on every continentincluding Africans, Asians, Australianaborigines, Caucasians, Peruvian, Mexican, Native Americans.

Links between porphyrias and mental illness have been noted fordecades. In the early 1950s patients with porphyrias and severesymptoms of depression were treated with electroshock.

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suggested as an explanation for the originof vampire and werewolf legends, based upon certain perceivedsimilarities between the condition and the folklore.

about eight different types of porphyria have been observed up to date.

The most recent trend of treatment is using Heme Therapy whereinHeme associated drugs are administered to the patient.

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Eminent personalities who suffered from Porphyria

Vincent Willem Van Gogh (painter) King George III (Britain)

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How is Porphyria classified?

Classification systems are based on :

1. Specific enzyme deficiency. For example : ALA Dehydratasedeficiency

2. Neurologic symptoms caused by the Porphyrias (acute porphyrias).For example : Hereditary coproporphyria,Acute intermittent porphyria

3. Photosensitivity (cutaneous porphyrias). For example : Porphyriacutanea tarda, variegate porphyria

4. whether the excess precursors originate primarily in the liver (hepaticporphyrias) or primarily in the bone marrow (erythropoieticporphyrias).

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Porphyria may lead to mental illness because :

Excess amounts of PBG or ALA may cause neurotoxicity.

Heme deficiency may result in degenerative changes in the centralnervous system.

Increased ALA concentrations in the brain may inhibit gamma-aminobutyric acid release(GABA).

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Clinical symptoms

1. Abdominal pain or cramping (Acute Porphyria)

2. Problems with the nervous system and muscles, seizures, mentaldisturbances, nerve damage (Acute Porphyria)

3. Light sensitivity causing rashes, blistering, and scarring of the skin

(Porphyria Cutanea tarda)

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Other symptoms may include:

Muscle pain

Muscle weakness or paralysis

Numbness or tingling

Pain in the arms or legs

Pain in the back

Personality changes

Low blood pressure

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Complications of the disease :

Coma or Paralysis

Respiratory failure (due to weakness of chest muscles)

Scarring of the skin

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Blisters as observed in Porphyria cutanea tarda patients

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Prevalence and StatisticsAround the world The exact prevalence of porphyria is unknown, but it probably ranges

from 1 in 500 to 1 in 50,000 people worldwide. (until 2011)

For some forms of porphyria, the prevalence is unknown because manypeople with a genetic mutation associated with the disease neverexperience signs or symptoms.

Acute intermittent porphyria (AIP) may occur more frequently innorthern European countries, such as Sweden, in the United Kingdomand in India.

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Hereditary coproporphyria, has been reported mostly in Europe and

North America.

Variegate porphyria is most common in the Afrikaner population ofSouth Africa; about 3 in 1,000 people in this population have thegenetic change that causes this form of the disorder.

Data obtained courtesy of-http://ghr.nlm.nih.gov/condition=porphyria ; service of USNational Library of Medicine.

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India

In India, AIP has been reported from various parts of the country andsome specific communities have been found to be especiallysusceptible.

Fifteen patients of AIP were detected out of2,500 persons of theMaheshwari communitysurveyed in Sri-Dungargarh municipal areaof Bikaner. The fifteen patients belonged to 11 families and the femaleto male ratio was 9:6. Most of them were encountered in the age group11 -20 years.

(Rajashekar Reddi, Nitin K Sethi, Ish Anand, PK Sethi., 2002,Acute IntermittentPorphyria : Management Aspects., JIACM, J Assoc Physicians India)

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Genetic Basis Each form of porphyria results from mutations in one of these

genes:ALAD,ALAS2, CPOX, FECH, HMBS, PPOX, UROD, or UROS.

1. These genes are necessary for coding for the enzymes needed toproduce heme.

2. The mutations reduce enzyme activity, which limits the amount ofheme produced.

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3. porphyrins and porphyrin precursors, formed during the processof heme production, accumulate in liver and other organs. Onaccumulation in the skin, the precursors interact with sunlight andcause the cutaneous forms of porphyria.

4. The acute forms of the disease occur when porphyrins and

porphyrin precursors build up in and damage the nervous system.

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Type Inheritance Gene Gene Locus Enzyme

ALA-Dehydratase

Porphyria (ADP)

Autosomal recessive ALAD 9q33.1 ALA dehydratase

Acute Intermittent

Porphyria (AIP)

Autosomal dominant HMBS 11q23.3 PBG deaminase

Congenital

Erythropoietic

Porphyria (CEP)

Autosomal recessive UROS 10q25.2-q26.3 Uroporphyrinogen III

synthase

Porphyria Cutanea

Tarda (PCT), familial

form

Autosomal dominant UROD 1p34 Uroporphyrinogen III

decarboxylase

Hepatoerythropoietic

Porphyria (HEP)

Autosomal recessive UROD 1p34 Uroporphyrinogen III

decarboxylase

Hereditary

Coproporphyria

(HCP)

Autosomal dominant CPOX 3q12 Coproporphyrinogen

III oxidase

Variegate Porphyria

(VP)

Autosomal dominant PPOX 1q22 Protoporphyrinogen

oxidase

Erythropoietic

Protoporphyria (EPP)

Autosomal recessive FECH 18q21.3 Ferrochelatase

X-linked

Protoporphyria

(XLP)

X-linked ALAS2 Xp11.21 ALA synthase

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Note : One type of porphyria, porphyria cutanea tarda, results from bothgenetic and nongenetic factors. About 20 percent of cases are related to

mutations in the UROD gene. The remaining cases are not associatedwith UROD gene mutations and are classified as sporadic. Factorscontributing to the development of porphyria cutanea tarda are :

1. An increased amount of Iron in the liver. Mutations in the HFEgene (which

cause an iron overload disorder) are also associated with porphyria cutaneatarda.

2. Hepatitis C or HIV infection.

3. Other, as-yet-unidentified genetic factors may also play a role in this formof porphyria.

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Representation of loci of genes involved in some common forms of Porphyria

short arm of chromosome 1; position 34 PCT (UroD gene)

long arm of chromosome 11; position 23.3 AIP (HMBS gene)

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Molecular BasisAcute Intermittent Porphyria

It is a hepatic porphyria.

Characterized by a deficiency of the enzyme porphobilinogendeaminase (PBGD) also known as hydroxymethylbilane synthase(HMBS).

Over 200 PBGD mutations have been reported worldwide, includingsmall insertions and deletions, missense mutations, as well asmutations in the PBGD promoter region

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HMBS/PBGD gene

comprises 8,673 bases. 15 exons.

There are 2 types of transcripts : Housekeeping and Erythroid-specific :formed by alternate splicing.

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Type I mutations Patients exhibit reduced enzymatic activity as well as reduced PBGDprotein content, both at approximately 50 percent of normal. Single

base substitutions or deletions leading to a single amino acid change,resulting in the loss of expression of the enzyme protein.

Type IImutations

They are characterized by decreased PBGD activity in nonerythroidcells (eg liver) at approximately 50 percent of normal, but with normalerythroid PBGD activity. single base substitutions which occur in theexon/intron boundary of exon 1

Type IIImutations

Occurs in exons 10 and 12, which are regions thought to be essential forcatalytic activity of the enzyme

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Diagnosis

Biochemical Diagnosis

Blood tests can be done to measure Porphyrin levels

Urinalysis

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Urinalysis

Urinalysis is the physical, chemical, and microscopic examination ofurine. It involves a number of tests to detect and measure variouscompounds that pass through the urine.

A special stick ("dipstick") tests for various substances in the urine. The

stick contains little pads of chemicals that change color when theycome in contact with the substances of interest. It is used to detectpresence of porphyrin precursors.

Upon further chemcical analysis and quantification, it has been foundthat :

Aminolevulinic acid Normal urinary excretion of ALA is

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Blood Test (Porphyrin level)

A blood sample is needed.

Three porphyrins can normally be measured in small amounts inhuman blood. They are:

1. Coproporphyrin

2. Protoporphyrin

3. Uroporphyrin

Protoporphyrin is normally found in the highest amount. More tests areneeded to show the levels of specific porphyrins.

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This test specifically measures total porphyrin levels, but referencevalues (a range of values seen in a group of healthy people) for theindividual components are also included:

Total porphyrin levels: 16 to 60 mcg/dL

Coproporphyrin levels: < 2 mcg/dL

Protoporphyrin levels: 16 to 60 mcg/dL

Uroporphyrin levels: < 2 mcg/dL

Note: mcg/dL = micrograms per deciliter

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What Abnormal Results Mean

Increased levels of coproporphyrins may be a sign of:

Hepatic coproporphyria

Increased protoporphyrin levels may be a sign of:

Congenital erythropoietic protoporphyria

Variegate porphyria

Increased uroporphyrin levels may be a sign of:

Porphyria cutanea tarda

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Molecular Diagnosis

Acute intermittent porphyria (AIP) is an autosomal dominant disordercaused by a porphobilinogen (PBG) deaminase deficiency. Adenaturing gradient gel electrophoresis (PCR-DGGE) analysis followed

by direct sequencing of the DNA fragments can be performed toinvestigate molecular defect.

According to this, sensitivity of the DGGE screening method was closeto 100%.

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What is Denaturing Gel Electrophoresis (DGGE)?

Denaturing gradient gels are used to detect mutations.

The genomic fragments are run on a low to high denaturant gradientacrylamide gel; initially the fragments move according to molecular

weight, but as they progress into higher denaturing conditions, each(depending on its sequence composition) reaches a point where the

DNA begins to melt and forces DNA molecules to unwind

The melting severely retards the progress of the molecule in the gel,and a mobility shift is observed.

It is the mobility shift which can differ for slightly different sequences(depending on the sequence, as little as a single bp change can cause amobility shift). Alleles are detected by differences in mobility. This canalso be used to differentiate diseased condition from normal usingknown genes responsible for a particular disease.

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Treatment

Note : Porphyria does not kill usually. It affects the quality of life.

Some of the medicines used to treat porphyria may include:

Heme therapy Panhematin (US), Heme-arginate.{Hematin administered intravenously while Panhematin is consumed;

form of alkaline heme; corrects heme deficiency in liver, repressesproduction of precursors e.g., decreases level of PBG in urine(important in AIP)}.

Propranolol to control the heartbeat as the heartbeat may get affectedalong with blood pressure in some cases of Porhyria.

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Other treatments may include:

Chloroquine - famous for use in malaria treatment, has also beenobserved to regulate ALA Synthase activity, causes reduction inprecursor formation and accumulation.

Fluids and glucose to boost carbohydrate levels, which helps limit theproduction of porphyrins.

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Depending on the type of porphyria, the doctor may suggest to take thefollowing preventive measures to control the discomfort and

symptoms:

Avoid alcohol

Avoid injuring the skin

Avoid sunlight as much as possible and use sunscreen when outside

Eat a high-carbohydrate diet

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An interesting fact to note :

Research studies have been done on mice models of CongenitalErythropoietic Porphyria for Gene Therapy.

The UROS gene is located on chromosome 10.

codes for uroporphyrinogen synthase (the 4th enzyme in the pathway).

Lentivirus-mediated transfer of the human UROS cDNA intohematopoietic stem cells (HSCs) from Uros(mut248) mice was done.

complete and long-term enzymatic, metabolic, and phenotypiccorrection of the disease, favoured by a survival advantage of correctedred blood cells.

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Prevention of the disease :

Genetic counselling may benefit people who want to

have children and who have a family history of anytype of Porphyria as most of them are geneticallyinherited.

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Current Research

It was observed that inhibition of delta-aminolevulinic acid synthase

led to hepatic heme depletion via induction or suicidal inactivation of

cytochrome P450. The process is now sufficiently understood such that

prediction of porphyria from structural and functional information of

the enzymes in an individual may be possible in the near future.

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Foundations and Associations

Around the World

American Porphyria Foundation: Laboratory Testing forPorphyria [porphyriafoundation.com]

The British Porphyria Association [porphyria.org.uk]

European Porphyria Initiative [irondisorders.org]

Iron Overload Diseases Association [ironoverload.org]

American Liver Foundation [liverfoundation.org]

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Case Reports and Institutes in INDIA doing research on Porphyria

J.L.N. Medical College & Hospital, Ajmer.

C. Kothari Medical & Research Institute, Bikaner, Rajasthan.

S.P. Medical College, Bikaner, Rajasthan.

Silchar Medical College, Silchar, Assam.

Bankura Sammilani Medical College, West Bengal.

R.G. Kar Medical College, Kolkata, West Bengal.

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Thank You